Vaccination and immunization generally refer to the introduction of a non-virulent agent against which an individual's immune system can initiate an immune response which will then be available to defend against challenge by a pathogen. The immune system identifies invading “foreign” compositions and agents primarily by identifying proteins and other large molecules which are not normally present in the individual. The foreign protein represents a target against which the immune response is made.
The immune system can provide multiple means for eliminating targets that are identified as foreign. These means include humoral and cellular responses which participate in antigen recognition and elimination. Briefly, the humoral response involves B cells which produce antibodies that specifically bind to antigens. There are two arms of the cellular immune response. The first involves helper T cells which produce cytokines and elicit participation of additional immune cells in the immune response. The second involves killer T cells, also known as cytotoxic T lymphocytes (CTLs), which are cells capable of recognizing antigens and attacking the antigen including the cell or particle it is attached to.
Vaccination has been singularly responsible for conferring immune protection against several human pathogens. In the search for safe and effective vaccines for immunizing individuals against infective pathogenic agents such as viruses, bacteria, and infective eukaryotic organisms, several strategies have been employed thus far. Each strategy aims to achieve the goal of protecting the individual against pathogen infection by administering to the individual, a target protein associated with the pathogen which can elicit an immune response. Thus, when the individual is challenged by an infective pathogen, the individual's immune system can recognize the protein and mount an effective defense against infection. There are several vaccine strategies for presenting pathogen proteins which include presenting the protein as part of a non-infective or less infective agent or as a discreet protein composition.
One strategy for immunizing against infection uses killed or inactivated vaccines to present pathogen proteins to an individual's immune system. In such vaccines, the pathogen is either killed or otherwise inactivated using means such as, for example, heat or chemicals. The administration of killed or inactivated pathogen into an individual presents the pathogen to the individual's immune system in a noninfective form and the individual can thereby mount an immune response against it. Killed or inactivated pathogen vaccines provide protection by directly generating T-helper and humoral immune responses against the pathogenic immunogens. Because the pathogen is killed or otherwise inactivated, there is little threat of infection.
Another method of vaccinating against pathogens is to provide an attenuated vaccine. Attenuated vaccines are essentially live vaccines which exhibit a reduced infectivity. Attenuated vaccines are often produced by passaging several generations of the pathogen through a permissive host until the progeny agents are no longer virulent. By using an attenuated vaccine, an agent that displays limited infectivity may be employed to elicit an immune response against the pathogen. By maintaining a certain level of infectivity, the attenuated vaccine produces a low level infection and elicits a stronger immune response than killed or inactivated vaccines. For example, live attenuated vaccines, such as the poliovirus and smallpox vaccines, stimulate protective T-helper, T-cytotoxic, and humoral immunities during their nonpathogenic infection of the host.
Another means of immunizing against pathogens is provided by recombinant vaccines. There are two types of recombinant vaccines: one is a pathogen in which specific genes are deleted in order to render the resulting agent non-virulent. Essentially, this type of recombinant vaccine is attenuated by design and requires the administration of an active, non-virulent infective agent which, upon establishing itself in a host, produces or causes to be produced antigens used to elicit the immune response. The second type of recombinant vaccine employs infective non-virulent vectors into which genetic material that encode target antigens is inserted. This type of recombinant vaccine similarly requires the administration of an active infective non-virulent agent which, upon establishing itself in a host, produces or causes to be produced, the antigen used to elicit the immune response. Such vaccines essentially employ infective non-virulent agents to present pathogen antigens that can then serve as targets for an anti-pathogen immune response. For example, the development of vaccinia as an expression system for vaccination has theoretically simplified the safety and development of infectious vaccination strategies with broader T-cell immune responses.
Another method of immunizing against infection uses subunit vaccines. Subunit vaccines generally consist of one or more isolated proteins derived from the pathogen. These proteins act as target antigens against which an immune response may be mounted by an individual. The proteins selected for subunit vaccine are displayed by the pathogen so that upon infection of an individual by the pathogen, the individuals immune system recognizes the pathogen and mounts a defense against it. Because subunit vaccines are not whole infective agents, they are incapable of becoming infective. Thus, they present no risk of undesirable virulent infectivity that is associated with other types of vaccines. It has been reported that recombinant subunit vaccines such as the hepatitis B surface antigen vaccine (HBsAg) stimulate a more specific protective T-helper and humoral immune response against a single antigen. However, the use of this technology to stimulate board protection against diverse pathogens remains to be confirmed.
The construction of effective vaccines is complicated by several factors which include the pathobiology of the pathogen and the specificities of the of the host immune response. Recently a novel tool for understanding the immune component in these interactions has become available in the form of genetic immunization or DNA vaccination. Tang, et al., Nature, 1992, 356, 152; Fynan, et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 11478; Ulmer, et al., Science, 1993, 259, 1745; and Wang, et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 4156. The ability of this approach was demonstrated to produce broad immune responses against structural and enzymatic gene products of HIV-1 and outlined a strategy for development of a possible prophylactic vaccine for HIV-1. This strategy utilized multiple gene expression cassettes encoding gag/pol/rev as well as env/rev and accessory gene immunogens. Studies clearly demonstrated that rodents and primates can be successfully immunized with HIV-1 structural and envelope genes. Wang, et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 4156 and Wang, et al., DNA Cell Biol., 1993, 12, 799. A genetic strategy for construction of immunogen expression cassettes from a pathogenic gene which can be broadly applied in order to use DNA immunogens against a variety of pathogens is needed.
Primate lentiviral genomes contain genes encoding novel regulatory and accessory proteins as well as proteins with structural and enzymatic functions. The regulatory genes, tat and rev, and the accessory genes, nef, vif, vpr, vpu, and vpx, are well conserved in many lentiviruses, including HIV and SIV. The well conserved nature of these genes implies that their protein products play a critical role in viral pathogenesis in vivo. Initial in vitro experiments seemed to demonstrate that tat and rev were essential for viral replication, while the accessory genes were considered nonessential. Cullen, et al., Cell, 1989, 58, 423 and Desrosiers, AIDS Res. Human Retroviruses, 1992, 8, 411. Further analyses, however, has revealed that defects within the accessory gene result in severe impairment or delay in viral replication in vitro (Gabudza, et al, J. Virol., 1992, 66, 6489 and Gibbs, et al., AIDS Res. Human Retroviruses, 1994, 10, 343) and in vivo (Aldrovandi, et al., J. Virol., 1996, 70, 1505). Native defective accessory genes have been reported in vivo and may be an end product of an effective host immune response. The accessory genes are, therefore, presently considered to be determinants of virus virulence. Trono, Cell, 1995, 82, 189. They contain few “hot spots” and may be less susceptible to mutations leading to the production of “escape” virus variants, emphasizing their importance in the viral life cycle. In addition, the protein products of these genes are immunogenic in vivo. As a group, they represent twenty percent of the possible anti-viral immune targets. Ameisen, et al., Int. Conf. AIDS, 1989, 5, 533 and Lamhamedi-Cherradi, et al., AIDS, 1992, 6, 1249. Their immunogenicity and low functional mutagenicity combine to make the accessory genes attractive elements in the design of future anti-viral immune therapeutics. The production of accessory gene immunogens poses specific immunologic and pathogenic complications for a viral vaccine design, however, due to the role of the accessory gene protein products as determinants of viral virulence. A potential accessory gene-based genetic vaccine would need to be accessible to the host's immune response against native viral accessory gene products without enhancing viral replication. Accordingly, a major goal is to design a safe and effective genetic anti-HIV vaccine, which includes the vif (virion infectivity factor) accessory gene as part of a multi-component genetic immunogen.
The vif gene encodes a 23 kDa late viral protein (vif) from a singly spliced, rev-dependent 5 kb transcript. Arya, et al., Proc. Natl. Acad. Sci. USA, 1986, 83, 2209; Garrett, et at, J. Virol., 1991, 65, 1653; Schwartz, et al., Virol., 1991, 183, 677; and Sodroski, et al., Science, 1986, 231, 1549. Vif is highly conserved among HIV-1 isolates and is present in other lentiviruses, such as Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), Visna virus, HIV-2, and SIV. Myers, et al., Human Retrovir. AIDS, 1991 and Shackett, et al., Virol., 1994, 204, 860. Earlier analyses of in vivo vif genetic variation have shown that most vif sequences are intact reading frames and the presence of intact vif does not have a correlation with disease status. Sova, et al, J. Virol., 1995, 69, 2557 and Wieland, et al., Virol., 1994, 203, 43. However, sequential analyses of a region containing vif, vpr, vpu, tat, and rev genes from a HIV-1 infected long-term progressor revealed the presence of inactivating mutations in 64% of the clones. Michael, et al., J. Virol., 1995, 69, 4228. HIV-1 infected subjects have been shown to carry antibodies which recognize recombinant vif protein (Kan, et al., Science, 1986, 231, 1553; Schwander, et al., J. Med. Virol., 1992, 36, 142; and Wieland, et al., AIDS Res. Human Retrovir., 1991, 7, 861) suggesting that the protein is expressed and is immunogenic during natural infection (Volsky, et al., Curr. Topics Micro. Immunol., 1995, 193, 157.
Due to vif's ability to activate viral replication in trans, an attenuated genetic vaccine design, similar to those utilized in the production of vaccines derived from toxic viral, bacterial, or parasitic components was employed in the present invention. The sequence variation and immunogenic potential present in vif genes derived from HIV-1 infected subjects was analyzed. Prototypic genetic variants were selected and the ability of those clones to induce humoral and cellular immune responses was studied in animals. The selected vif genetic variants were also functionally characterized through transcomplementation assays utilizing cells infected with a vif-defective HIV-1 clone. Attenuated, nonfunctional vif clones are demonstrated to induce immune responses capable of destroying native pathogen.